A growing number of diseases and syndromes are known to be associated with abnormalities in platelet number, function, or morphology. Classical platelet disorders, such as thrombocytopenia, have been joined by cardiac thrombosis, infectious pathologies and even Alzheimer's disease in the list of disorders associated with platelet abnormalities. See Gurbel, Am. Heart J., 139: 320-328 (2000); Tetta et al., Am. J. Kidney Dis., 30: S57-S65, (1997); Cohen et al., Life Sci., 40: 2445-2451 (1987).
In some cases, detection of the platelet abnormality may depend on determining the presence or absence of certain platelet surface antigens. For example, Glanzmann's Thrombasthenia is characterized by an absence of the GPIIb-IIIa complex (GPIIB/GPIIIA complex). Similarly, Bernard-Soulier syndrome is characterized by a deficiency in the GPIb-IX-V complex (CD42a-d). In other cases, the results of platelet functional tests, to evaluate the release of soluble markers and surface expression, are important. For example, the differential release of granule contents (such as CD62P and CD63) after various stimulation procedures may reflect deficiencies in the storage pools of the platelets.
As the number of associated pathologies grows, there is an increasing need to enumerate and characterize (i.e., “phenotype”) platelets. Unfortunately, current methods for assaying platelets have a number of drawbacks. Automated platelet counters are available that can accurately determine the number of platelets in a sample. However, platelet counters do not provide information about expressed surface antigens or activation state of the platelets.
Measuring platelet aggregation and platelet-monocyte aggregation also provides potentially valuable information. Aggregometers, which measure platelet aggregation in response to various stimuli, provide information about platelet function and can be used to assess the efficacy of antiplatelet therapy. However, the most common activation procedures do not allow for the detection of soluble released materials or the characterization of platelet surface antigens.
Flow cytometry can be used to obtain important phenotypic information about platelets. The platelets in a sample can be directly counted. In addition, platelet surface markers can be identified and characterized through the use of fluorophore-labeled antibodies against specific cell surface antigens. However, use of flow cytometry to study platelets is limited by the reactive nature of the platelets themselves. Platelets may artificially become activated by exposure to the high-pressure fluidics system. To address this problem, flow cytometry phenotyping procedures typically include a fixation step to preserve the platelet sample. Although fixation eliminates the possibility of artificial activation, it also precludes the ability to subsequently stimulate and measure platelet responsiveness. See Matzdorff et al., Laboratory Hematology 4, 163-168, Carden Jennings Publishing Co., Ltd. (1998); Rinder, Clin. Lab. Sci., 11: 365-72, (1998); Furman et al., J. Am. Coll. Cardiol., 31:352-358 (1998); Michelson, Blood Coagul.Fibrinolysis, 5:121-131 (1994); Becker et al., Coron. Artery Dis., 5:339-345 (1994).
Sample fixation may also interfere with antibody binding to platelets. It is known that PAC-1 will not bind to paraformaldehyde-fixed platelets and that CD62P binding is decreased. Additionally, many of the substances used for fixation are toxic and require special handling. Exposure to formaldehyde, for example, may cause cancer.
To prevent the blood from clotting, it is standard practice to use anticoagulants with whole blood samples. Ethylene-diamino-tetra-acetic acid (EDTA) is perhaps the most commonly used anticoagulant for hematologic studies. EDTA prevents coagulation by chelating calcium ions. The newer commercially available collection tube referred to as the CTAD tube (also known as Diatube H)—which includes the anticoagulant sodium citrate, and three inhibitors of platelet activation, theophylline, adenosine and dipyridamole—inhibits platelet activation. See Kuhne et al., Am. J. Hematol., 50:40-45 (1995); Mody et al., Transfus. Med., 9:147-154 (1999); O'Connor et al., Am. J. Cardiol., 83:1345-1349 (1999). Other combinations of platelet activation inhibitors may also be used. However, because of the forces involved in the fluidics of a cytometer, fixation is still recommended prior to examination on a cytometer. See Rinder et al. and Furman et al., supra.
In order to allow further activation studies, it is important to examine platelets in their “native” whole blood state without fixation. Thus, there is a need for an analytical procedure that allows activation and whole blood studies to be performed on the same sample. To measure both platelet surface antigens and soluble platelet activation products from the same sample, for example, would increase the total information content attainable from a sample.
Microvolume laser scanning cytometry (MLSC) provides a powerful method for monitoring fluorescently labeled cells in whole blood, processed blood, and other fluids. As with flow cytometry, fluorophore-labeled antibodies specific for cell surface antigens are used to identify, characterize and enumerate specific cell populations. The reaction can be done in whole blood and in general, there is no need to wash unbound reagent away. The cell-antibody mixture is loaded into an optical-quality capillary of known volume and analyzed with the laser-based fluorescence-imaging instrument. A small cylindrical laser spot is scanned across the stationary cells to produce an image of the entire capillary. Image-processing software is used to analyze the image and identify and enumerate the cells of interest. Because the interrogation volume of the capillary is known, absolute cell counts (cells per μl) are obtained. See, e.g., U.S. Pat. Nos. 5,547,849 and 5,556,764; Dietz et al., Cytometry 23: 177-186 (1996), each of which is incorporated herein by reference in its entirety. Importantly, such capillary cytometry eliminates the fluidic forces used in flow cytometry.
The SurroScan™ MLSC (SurroMed, Mountain View, Calif.) is able to classify and quantify hundreds of cell populations in small volumes of unprocessed whole blood and other biological fluids by detection of combinations of fluorophore-labeled antibodies to cell surface markers. See International Patent Application Serial No. PCT/US00/11133, entitled “Novel Optical Architectures for Microvolume Laser-Scanning Cytometers,” published Mar. 2, 2000; Walton et al., Proc. SPIE-Int. Soc. Opt. Eng., 3926:192-201(2000) IBOS Society of Photo-Optical Instrumentation Engineers, both incorporated herein by reference in their entirety.